Guanine Nucleotide Exchange Factor Tiam1 Research Articles

TIAM-1 regulates polarized protrusions during dorsal intercalation in the Caenorhabditis elegans embryo through both its GEF and N-terminal domains.

Mediolateral cell intercalation is a morphogenetic strategy used throughout animal development to reshape tissues. Dorsal intercalation in the Caenorhabditis elegans embryo involves the mediolateral intercalation of two rows of dorsal epidermal cells to create a single row that straddles the dorsal midline, and thus is a simple model to study cell intercalation. Polarized protrusive activity during dorsal intercalation requires the C. elegans Rac and RhoG orthologs CED-10 and MIG-2, but how these GTPases are regulated during intercalation has not been thoroughly investigated. In this study, we characterized the role of the Rac-specific guanine nucleotide exchange factor (GEF) TIAM-1 in regulating actin-based protrusive dynamics during dorsal intercalation. We found that TIAM-1 can promote formation of the main medial lamellipodial protrusion extended by intercalating cells through its canonical GEF function, whereas its N-terminal domains function to negatively regulate the generation of ectopic filiform protrusions around the periphery of intercalating cells. We also show that the guidance receptor UNC-5 inhibits these ectopic filiform protrusions in dorsal epidermal cells and that this effect is in part mediated via TIAM-1. These results expand the network of proteins that regulate basolateral protrusive activity during directed rearrangement of epithelial cells in animal embryos.

Rho5 regulates lysophosphatidic acid induced macropinocytosis in Entamoeba histolytica

Entamoeba histolytica is a unicellular enteric protozoan responsible for variety of gastrointestinal pathologies. The pathogenicity of the parasite is attributed to its dynamic cytoskeleton. Rho family GTPases, a major class of molecular switches, play an indispensable role in controlling cytoskeleton remodelling. Rho GTPases are known to be activated by specific Guanine nucleotide Exchange Factors (GEF) upon stimulation by various extracellular stimuli to regulate signalling.We have established that EhRho5 is important for LPA induced micropinocytosis in the amoebic trophozoites. EhRho5 translocated from cytosol to plasma membrane and endomembranous compartments when stimulated with LPA, a bioactive phospholipid known to activate Rho subfamily members (1,2). Our findings were strengthened when FRAP (Fluorescence recovery after photobleaching) study showed that LPA treated cells show 30% faster recovery of Rho5 when compared to serum starved cells. The translocation is accompanied by activation of the GTPase as revealed by effector pull down assay(2). LPA stimulation enhanced the macropinocytic efficiency of trophozoites in a Rho5 dependent manner. We then identified the GEF for Rho5 which too, shows translocation upon stimulation with LPA. Also, depletion of the GEF leads to decreased pinocytic efficiency of the trophozoites. Using inhibitor based studies, we further demonstrated that PI3K acts as an intermediate cue for EhRho5 activation during LPA induced macropinocytosis.Moreoever, Rho5 expression showed contrary relationship with random migration of the trophozoites suggesting a pivotal role of the GTPase in mutally antagonistic cellular processes(3) in the amoeba. Reference: Fleming IN, Elliott CM, Exton JH. Differential Translocation of Rho Family GTPases by Lysophosphatidic Acid, Endothelin‐1, and Platelet‐derived Growth Factor, J. Biol. Chem. 1996, 271: 33067–33073 Van Leeuwen FN, Olivo C, Grivell S, Giepmans BNG, Collard JG, Moolenaar WH. Rac Activation by Lysophosphatidic Acid LPA1 Receptors through the Guanine Nucleotide Exchange Factor Tiam1, J. Biol. Chem. 2003, 278:400–406 Veltman DM, Lemieux MG, Knecht DA, Insall RH. PIP3‐dependent macropinocytosis is incompatible with chemotaxis. Journal of Cell Biology. 2014 Feb 17;204(4):497‐505.

Identification of Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation in glioblastoma

The protein Lgl1 is a key regulator of cell polarity. We previously showed that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN tumour suppressor loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C that has been activated by binding to a complex of the scaffolding protein Par6 and active, GTP-bound Rac. The specific Rac guanine nucleotide exchange factors that generate active Rac to promote Lgl1 hyperphosphorylation in glioblastoma are unknown. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation, which was reversed by re-expressing PREX1. They also had reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. This was due to overexpression of a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in these PREX1 knockout cells reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 is also to fill this role in a subset of patients. This redundancy between PREX1 and TIAM1 is only partial, as motility was impaired in PREX1 knockout cells from both patients.

The RAC1 activator Tiam1 regulates centriole duplication through controlling PLK4 levels.

ABSTRACTCentriole duplication is tightly controlled to maintain correct centriole number through the cell cycle. Key to this is the regulated degradation of PLK4, the master regulator of centriole duplication. Here, we show that the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 localises to centrosomes during S-phase, where it is required for the maintenance of normal centriole number. Depletion of Tiam1 leads to an increase in centrosomal PLK4 and centriole overduplication, whereas overexpression of Tiam1 can restrict centriole overduplication. Ultimately, Tiam1 depletion leads to lagging chromosomes at anaphase and aneuploidy, which are potential drivers of malignant progression. The effects of Tiam1 depletion on centrosomal PLK4 levels and centriole overduplication can be rescued by re-expression of both wild-type Tiam1 and catalytically inactive (GEF*) Tiam1, but not by Tiam1 mutants unable to bind to the F-box protein βTRCP (also known as F-box/WD repeat-containing protein 1A) implying that Tiam1 regulates PLK4 levels through promoting βTRCP-mediated degradation independently of Rac1 activation.

Par3 is essential for the establishment of planar cell polarity of inner ear hair cells

In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3-GEF-Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.

TIAM-1/GEF can shape somatosensory dendrites independently of its GEF activity by regulating F-actin localization.

Dendritic arbors are crucial for nervous system assembly, but the intracellular mechanisms that govern their assembly remain incompletely understood. Here, we show that the dendrites of PVD neurons in Caenorhabditis elegans are patterned by distinct pathways downstream of the DMA-1 leucine-rich transmembrane (LRR-TM) receptor. DMA-1/LRR-TM interacts through a PDZ ligand motif with the guanine nucleotide exchange factor TIAM-1/GEF in a complex with act-4/Actin to pattern higher order 4° dendrite branches by localizing F-actin to the distal ends of developing dendrites. Surprisingly, TIAM-1/GEF appears to function independently of Rac1 guanine nucleotide exchange factor activity. A partially redundant pathway, dependent on HPO-30/Claudin, regulates formation of 2° and 3° branches, possibly by regulating membrane localization and trafficking of DMA-1/LRR-TM. Collectively, our experiments suggest that HPO-30/Claudin localizes the DMA-1/LRR-TM receptor on PVD dendrites, which in turn can control dendrite patterning by directly modulating F-actin dynamics through TIAM-1/GEF.

Peptide modulators of Rac1/Tiam1 protein-protein interaction: An alternative approach for cardiovascular diseases.

Rac1 GTPase interaction with guanine nucleotide exchange factor Tiam1 is involved in several cancer types and cardiovascular diseases. Although small molecules interfering with their protein-protein interaction (PPI) were identified and studied, the ability of small peptides and peptide mimics acting as Rac1/Tiam1 PPI inhibitors has not been yet explored. Using computational alanine scanning (CAS), the "hot" interfacial residues have been determined allowing the design of a small library of putative PPI inhibitors. In particular, the insertion of an unnatural alpha, alpha disubstituted amino acid, that is norbornane amino acid, and the side chain stapling have been evaluated regarding both conformational stability and biological activity. REMD calculations and CD studies have indicated that one single norbornane amino acid at the N-terminus is not sufficient to stabilize the helix structure, while the side-chain stapling is a more efficient strategy. Furthermore, both engineered peptides have been found able to reduce Rac1-GTP levels in cultured human smooth muscle cells, while wild type sequence is not active.

Tiam1 promotes thyroid carcinoma metastasis by modulating EMT via Wnt/β-catenin signaling

Aberrant expression of the guanine nucleotide exchange factor Tiam1 is implicated in the invasive phenotype of many cancers. However, its involvement in thyroid carcinoma and downstream molecular events remains largely undefined. Here, we examined the effects of Tiam1 on the invasiveness and metastasis of thyroid carcinoma in vitro and in vivo and explored the underlying mechanisms by investigating the regulation of Tiam1 expression and the downstream pathways affected. Our results showed that Tiam1 knockdown inhibited the migratory and invasive capacity of thyroid cancer cells, suppressed epithelial-mesenchymal transition (EMT), and inhibited Wnt/β-catenin signaling in vitro. Moreover, Tiam1 knockdown suppressed liver metastasis development in vivo. The effects of Tiam1 on metastasis and EMT mediated by the Wnt/β-catenin pathway were reversed by Rac1 silencing, suggesting that the prometastatic effect of Tiam1 is mediated by the activation of Rac1. These results indicate that Tiam1 may be a prognostic factor and potential therapeutic target for the treatment of thyroid cancers.

Differential regulation of the Rac1 GTPase–activating protein (GAP) BCR during oxygen/glucose deprivation in hippocampal and cortical neurons

Brain ischemia causes oxygen and glucose deprivation (OGD) in neurons, triggering a cascade of events leading to synaptic accumulation of glutamate. Excessive activation of glutamate receptors causes excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts, but the mechanisms that underlie this difference are unclear. Signaling via Rho-family small GTPases, their upstream guanine nucleotide exchange factors, and GTPase-activating proteins (GAPs) is differentially dysregulated in response to OGD/ischemia in hippocampal and cortical neurons. Increased Rac1 activity caused by OGD/ischemia contributes to neuronal death in hippocampal neurons via diverse effects on NADPH oxidase activity and dendritic spine morphology. The Rac1 guanine nucleotide exchange factor Tiam1 mediates an OGD-induced increase in Rac1 activity in hippocampal neurons; however, the identity of an antagonistic GAP remains elusive. Here we show that the Rac1 GAP breakpoint cluster region (BCR) associates with NMDA receptors (NMDARs) along with Tiam1 and that this protein complex is more abundant in hippocampal compared with cortical neurons. Although total BCR is similar in the two neuronal types, BCR is more active in hippocampal compared with cortical neurons. OGD causes an NMDAR- and Ca2+-permeable AMPAR-dependent deactivation of BCR in hippocampal but not cortical neurons. BCR knockdown occludes OGD-induced Rac1 activation in hippocampal neurons. Furthermore, disrupting the Tiam1–NMDAR interaction with a fragment of Tiam1 blocks OGD-induced Tiam1 activation but has no effect on the deactivation of BCR. This work identifies BCR as a critical player in Rac1 regulation during OGD in hippocampal neurons.

Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity.

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens.

Neuronal Apoptosis Induced by Selective Inhibition of Rac GTPase versus Global Suppression of Rho Family GTPases Is Mediated by Alterations in Distinct Mitogen-activated Protein Kinase Signaling Cascades

Rho family GTPases play integral roles in neuronal differentiation and survival. We have shown previously that Clostridium difficile toxin B (ToxB), an inhibitor of RhoA, Rac1, and Cdc42, induces apoptosis of cerebellar granule neurons (CGNs). In this study, we compared the effects of ToxB to a selective inhibitor of the Rac-specific guanine nucleotide exchange factors Tiam1 and Trio (NSC23766). In a manner similar to ToxB, selective inhibition of Rac induces CGN apoptosis associated with enhanced caspase-3 activation and reduced phosphorylation of the Rac effector p21-activated kinase. In contrast to ToxB, caspase inhibitors do not protect CGNs from targeted inhibition of Rac. Also dissimilar to ToxB, selective inhibition of Rac does not inhibit MEK1/2/ERK1/2 or activate JNK/c-Jun. Instead, targeted inhibition of Rac suppresses distinct MEK5/ERK5, p90Rsk, and Akt-dependent signaling cascades known to regulate the localization and expression of the Bcl-2 homology 3 domain-only protein Bad. Adenoviral expression of a constitutively active mutant of MEK5 is sufficient to attenuate neuronal cell death induced by selective inhibition of Rac with NSC23766 but not apoptosis induced by global inhibition of Rho GTPases with ToxB. Collectively, these data demonstrate that global suppression of Rho family GTPases with ToxB causes a loss of MEK1/2/ERK1/2 signaling and activation of JNK/c-Jun, resulting in diminished degradation and enhanced transcription of the Bcl-2 homology 3 domain-only protein Bim. In contrast, selective inhibition of Rac induces CGN apoptosis by repressing unique MEK5/ERK5, p90Rsk, and Akt-dependent prosurvival pathways, ultimately leading to enhanced expression, dephosphorylation, and mitochondrial localization of proapoptotic Bad.

Differential Tiam1/Rac1 Activation in Hippocampal and Cortical Neurons Mediates Differential Spine Shrinkage in Response to Oxygen/Glucose Deprivation

Distinct neuronal populations show differential sensitivity to global ischemia, with hippocampal CA1 neurons showing greater vulnerability compared to cortical neurons. The mechanisms that underlie differential vulnerability are unclear, and we hypothesize that intrinsic differences in neuronal cell biology are involved. Dendritic spine morphology changes in response to ischemic insults in vivo, but cell type-specific differences and the molecular mechanisms leading to such morphologic changes are unexplored. To directly compare changes in spine size in response to oxygen/glucose deprivation (OGD) in cortical and hippocampal neurons, we used separate and equivalent cultures of each cell type. We show that cortical neurons exhibit significantly greater spine shrinkage compared to hippocampal neurons. Rac1 is a Rho-family GTPase that regulates the actin cytoskeleton and is involved in spine dynamics. We show that Rac1 and the Rac guanine nucleotide exchange factor (GEF) Tiam1 are differentially activated by OGD in hippocampal and cortical neurons. Hippocampal neurons express more Tiam1 than cortical neurons, and reducing Tiam1 expression in hippocampal neurons by shRNA enhances OGD-induced spine shrinkage. Tiam1 knockdown also reduces hippocampal neuronal vulnerability to OGD. This work defines fundamental differences in signalling pathways that regulate spine morphology in distinct neuronal populations that may have a role in the differential vulnerability to ischemia.

Hepatocyte growth factor triggers distinct mechanisms of Asef and Tiam1 activation to induce endothelial barrier enhancement

Previous reports described an important role of hepatocyte growth factor (HGF) in mitigation of pulmonary endothelial barrier dysfunction and cell injury induced by pathologic agonists and mechanical forces. HGF protective effects have been associated with Rac-GTPase signaling pathway activated by Rac-specific guanine nucleotide exchange factor Tiam1 and leading to enhancement of intercellular adherens junctions. This study tested involvement of a novel Rac-specific activator, Asef, in endothelial barrier enhancement by HGF and investigated a mechanism of HGF-induced Asef activation. Si-RNA-based knockdown of Tiam1 and Asef had an additive effect on attenuation of HGF-induced Rac activation and endothelial cell (EC) barrier enhancement. Tiam1 and Asef activation was abolished by pharmacologic inhibitors of HGF receptor and PI3-kinase. In contrast to Tiam1, Asef interacted with APC and associated with microtubule fraction upon HGF stimulation. EC treatment by low dose nocodazole to inhibit peripheral microtubule dynamics partially attenuated HGF-induced Asef peripheral translocation, but had negligible effect on Tiam1 translocation. These effects were associated with attenuation of HGF-induced barrier enhancement in EC pretreated with low ND dose and activation of Rac and its cytoskeletal effectors PAK1 and cortactin. These data demonstrate, that in addition to microtubule-independent Tiam1 activation, HGF engages additional microtubule- and APC-dependent pathway of Asef activation. These mechanisms may complement each other to provide the fine tuning of Rac signaling and endothelial barrier enhancement in response to various agonists.

Tiam1/Rac1 signals contribute to the proliferation and chemoresistance, but not motility, of chronic lymphocytic leukemia cells

AbstractSignals from the tumor microenvironment promote the migration, survival, and proliferation of chronic lymphocytic leukemia (CLL) cells. Rho GTPases control various signaling pathways downstream of microenvironmental cues. Here, we analyze the function of Rac1 in the motility and proliferation of CLL cells. We found decreased transcription of the Rac guanine nucleotide exchange factors Tiam1 and Vav1 in unstimulated peripheral blood CLL cells with almost complete loss of Tiam1 but increased transcription of the potential Rac antagonist RhoH. Consistently, stimulation of CLL cells with the chemokine CXCL12 induced RhoA but not Rac1 activation, whereas chemokine-induced CLL cell motility was Rac1-independent. Coculture of CLL cells with activated T cells induced their activation and subsequent proliferation. Here, Tiam1 expression was induced in the malignant cells in line with increased Ki-67 and c-Myc expression. Rac1 or Tiam1 knockdown using siRNA or treatment with the Tiam1/Rac inhibitor NSC-23766 attenuated c-Myc transcription. Furthermore, treatment of CLL cells with NSC-23766 reduced their proliferation. Rac inhibition also antagonized the chemoresistance of activated CLL cells toward fludarabine. Collectively, our data suggest a dynamic regulation of Rac1 function in the CLL microenvironment. Rac inhibition could be of clinical use by selectively interfering with CLL cell proliferation and chemoresistance.

Rap-afadin axis in control of Rho signaling and endothelial barrier recovery

Activation of the Rho GTPase pathway determines endothelial cell (EC) hyperpermeability after injurious stimuli. To date, feedback mechanisms of Rho down-regulation critical for barrier restoration remain poorly understood. We tested a hypothesis that Rho down-regulation and barrier recovery of agonist-stimulated ECs is mediated by the Ras family GTPase Rap1. Thrombin-induced EC permeability driven by rapid activation of the Rho GTPase pathway was followed by Src kinase-dependent phosphorylation of the Rap1-specific guanine nucleotide exchange factor (GEF) C3G, activation of Rap1, and initiation of EC barrier recovery. Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution. Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin. This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps. These data demonstrate that activation of the Rap1-afadin axis is a physiological mechanism driving restoration of barrier integrity in agonist-stimulated EC monolayers via negative-feedback regulation of Rho signaling, stimulation of actin peripheral dynamics, and reestablishment of cell-cell adhesive complexes.

Abstract A86: SIRT1 regulates Rac1-GTPase activation via the guanine nucleotide exchange factor TIAM1

Abstract SIRT1 deacetylase is overexpressed in different types of cancers and we and a few others have described a role for SIRT1 in regulating cancer cell migration. Rho GTPases are classic regulators of cell motility and are known to aberrantly enhance cancer cell migration in response to several anomalous cues. We hypothesized that one of mechanisms employed by SIRT1 to regulate cell motility is through regulation of Rac1-GTPase activation. To address this we utilized two cancer cell lines, CFPAC-1 pancreatic and MDA-MB-231 breast cancer cells, as our model. A significant decrease was observed in active Rac1 (GTP-bound Rac1) levels, in a GST-PAK-binding domain pull-down assay, upon inhibition of SIRT1 by small molecule inhibitors or knockdown of SIRT1 by shRNA as compared to vehicle-treated or non-targeting shRNA transfected cells. Subsequent analysis revealed that SIRT1 co-immunoprecipitated with the Rac-guanine nucleotide exchange factor (GEF), TIAM1. To test whether TIAM1 is acetylated and if SIRT1 regulates its deacetylation we performed antibody-based pull-down of acetylated lysines from whole cell protein extracts and observed TIAM1 acetylation. Inhibition of deacetylase activity by small molecule inhibitors or overexpression of acetyltransferases caused an enhancement of acetylated TIAM1 signal. While a change in TIAM1 localization was not observed with SIRT1 knockdown, its ability to activate Rac1 was compromised. Several studies have demonstrated SIRT1's pro-tumorigenic potential. In this study we elucidate a mechanism by which SIRT1 regulates Rac-GTPase activation via TIAM1 acetylation. Controlling the “switch-mechanism” that the small GTPases utilize for inducing downstream activity can be crucial from a drug-development perspective. Our findings not only provide a new mechanism for regulation of Rac-GTPase activity but also emphasize SIRT1's potential as a targetable molecule in cancer. This work is supported by CA155223 and the graduate stipend for Madhurima Saxena is provided by a Carroll-Feist Pre-doctoral Fellowship awarded by the Feist-Weiller Cancer Center at LSU Health Shreveport, Shreveport, LA. Citation Format: Madhurima Saxena, Samantha S. Dykes, Allison E. Wang, James A. Cardelli2,3, Kevin Pruitt1,3. SIRT1 regulates Rac1-GTPase activation via the guanine nucleotide exchange factor TIAM1. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A86.

Tiam1 increases turnover

![Figure][1] A control cell stained for actin (red) spreads out and polarizes on fibronectin (left), whereas a cell lacking Tiam1 remains compact and largely unpolarized (right). The guanine nucleotide exchange factor Tiam1 promotes cell migration by regulating the turnover of cell–

TrkB phosphorylation by Cdk5 is required for activity-dependent structural plasticity and spatial memory

The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB participate in diverse neuronal functions, including activity-dependent synaptic plasticity that is crucial for learning and memory. On binding to BDNF, TrkB is not only autophosphorylated at tyrosine residues but also undergoes serine phosphorylation at S478 by the serine/threonine kinase cyclin-dependent kinase 5 (Cdk5). However, the in vivo function of this serine phosphorylation remains unknown. We generated knock-in mice lacking this serine phosphorylation (Trkb(S478A/S478A) mice) and found that the TrkB phosphorylation-deficient mice displayed impaired spatial memory and compromised hippocampal long-term potentiation (LTP). S478 phosphorylation of TrkB regulates its interaction with the Rac1-specific guanine nucleotide exchange factor TIAM1, leading to activation of Rac1 and phosphorylation of S6 ribosomal protein during activity-dependent dendritic spine remodeling. These findings reveal the importance of Cdk5-mediated S478 phosphorylation of TrkB in activity-dependent structural plasticity, which is crucial for LTP and spatial memory formation.

VE‐cadherin trans‐interactions modulate Rac activation and enhancement of lung endothelial barrier by iloprost

Small GTPase Rac is important regulator of endothelial cell (EC) barrier enhancement by prostacyclin characterized by increased peripheral actin cytoskeleton and increased interactions between VE-cadherin and other adherens junction (AJ) proteins. This study utilized complementary approaches including siRNA knockdown, culturing in Ca(2+) -free medium, and VE-cadherin blocking antibody to alter VE-cadherin extracellular interactions to investigate the role of VE-cadherin outside-in signaling in modulation of Rac activation and EC barrier regulation by prostacyclin analog iloprost. Spatial analysis of Rac activation in pulmonary EC by FRET revealed additional spike in iloprost-induced Rac activity at the sites of newly formed cell-cell junctions. In contrast, disruption of VE-cadherin extracellular trans-interactions suppressed iloprost-activated Rac signaling and attenuated EC barrier enhancement and cytoskeletal remodeling. These inhibitory effects were associated with decreased membrane accumulation and activation of Rac-specific guanine nucleotide exchange factors (GEFs) Tiam1 and Vav2. Conversely, plating of pulmonary EC on surfaces coated with extracellular VE-cadherin domain further promoted iloprost-induced Rac signaling. In the model of thrombin-induced EC barrier recovery, blocking of VE-cadherin trans-interactions attenuated activation of Rac pathway during recovery phase and delayed suppression of Rho signaling and restoration of EC barrier properties. These results suggest that VE-cadherin outside-in signaling controls locally Rac activity stimulated by barrier protective agonists. This control is essential for maximal EC barrier enhancement and accelerated barrier recovery.

A novel player in cellular hypertrophy: Giβγ/PI3K-dependent activation of the RacGEF TIAM-1 is required for α1-adrenoceptor induced hypertrophy in neonatal rat cardiomyocytes

Activation of α(1)-adrenoceptors (α(1)-AR) by high catecholamine levels, e.g. in heart failure, is thought to be a driving force of cardiac hypertrophy. In this context several downstream mediators and cascades have been identified to potentially play a role in cardiomyocyte hypertrophy. One of these proteins is the monomeric G protein Rac1. However, until now it is unclear how this essential G protein is activated by α(1)-AR agonists and what are the downstream targets inducing cellular growth. By using protein-based as well as pharmacological inhibitors and the shRNA technique, we demonstrate that in neonatal rat cardiomyocytes (NRCM) Rac1 is activated via a cascade involving the α(1A)-AR subtype, G(i)βγ, the phosphoinositide-3'-kinase and the guanine nucleotide exchange factor Tiam1. We further demonstrate that this signaling induces an increase in protein synthesis, cell size and atrial natriuretic peptide expression. We identified the p21-activated kinase 2 (PAK2) as a downstream effector of Rac1 and were able to link this cascade to the activation of the pro-hypertrophic kinases ERK1/2 and p90RSK. Our data thus reveal a prominent role of the α(1A)-AR/G(i)βγ/Tiam1-mediated activation of Rac1 and its effector PAK2 in the induction of hypertrophy in NRCM.